EP1504123A4 - Methods for discovering tumor biomarkers and diagnosing tumors - Google Patents
Methods for discovering tumor biomarkers and diagnosing tumorsInfo
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- EP1504123A4 EP1504123A4 EP03726583A EP03726583A EP1504123A4 EP 1504123 A4 EP1504123 A4 EP 1504123A4 EP 03726583 A EP03726583 A EP 03726583A EP 03726583 A EP03726583 A EP 03726583A EP 1504123 A4 EP1504123 A4 EP 1504123A4
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- Prior art keywords
- cancer
- mammal
- biomarker
- prostate
- cancer cells
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
Definitions
- the present invention relates generally to genes useful as diagnostic markers and/or targets for therapeutic intervention in cancers. More particularly, the present invention concerns the identification of genes that encode secreted proteins and are differentially expressed in malignant and normal tissues. Methods are provided for the diagnosis, prognosis and treatment of various cancers based upon these genes.
- Cancer is a leading cause of death in the United States, causing one in four deaths, which is second only to heart disease. More than half a million people die of cancer each year in the United States. Four cancer sites, the lung, prostate, breast and colon, account for 56 percent of all new cancer cases and are the leading causes of cancer deaths for every racial and ethnic group, according to the Annual Report to the National on the Status of Cancer, 1973-1998 (Howe et al. (2001) J. Nat 'I. Cancer Institute 93: 824-842). The early stages of these and other types of cancer are often curable by, for example, surgery, radiation therapy or chemotherapy. Accordingly, early diagnosis of cancer is critical for effective treatment.
- PSA prostate-specific antigen
- PSA is specific for prostate tissue, it is produced by normal as well as malignant prostate tissue, and quantification of PSA expression in a fragment of prostate tissue does not unambiguously classify that tissue with respect to malignancy or malignant potential.
- PSA serum levels are an effective indicator of prostate cancer, modestly elevated levels, e.g., between 4 and 10 ng/mL are seen in men with obstructive or inflammatory uropathies, lowering the specificity of PSA as a cancer marker as described, e.g., in Brawer et al., Am. J. Clin. Pathol., Vol.
- biomarkers such as glandular kallikrein 2 (hK2) and prostate specific transglutaminase (pTGase), have been proposed as adjuncts to PSA to increase diagnostic specificity as described, e.g., in Nam et al., J Clin. Oncol., Vol. 18, pp. 1036- 1042 (2000), and reduce the number of men subjected to unnecessary biopsy, but the usefulness of these markers is still under investigation.
- hK2 glandular kallikrein 2
- pTGase prostate specific transglutaminase
- the invention provides methods for identifying a biomarker that is diagnostic for the presence of a cancer in a mammal.
- the methods involve: (a) analyzing one or more polynucleotide sequences using an algorithm that determines whether the polynucleotide sequence is predicted to encode a polypeptide that is secreted from a cell in which the polypeptide is expressed; and (b) determining whether an mRNA that corresponds to one or more of the polynucleotide sequences that are predicted to encode secreted polypeptides is differentially expressed in one or more types of cancer cells compared to non-cancer cells.
- An mRNA that is differentially expressed in cancer cells compared to non-cancer cells, or a polypeptide encoded by the differentially expressed mRNA is useful as a biomarker that is diagnostic for the presence of the cancer in a mammal.
- An mRNA that is differentially expressed in cancer cells compared to non-cancer cells, or a polypeptide encoded by the differentially expressed mRNA is useful as a biomarker that is diagnostic for the presence of the cancer in a mammal.
- the invention also provides methods for monitoring the efficacy of a cancer treatment in a mammal. These methods involve detecting an increase or decrease in the level of a biomarker that is diagnostic for the presence of the cancer in a mammal in a plurality of samples obtained from the mammal at different times, wherein the biomarker was identified using a method that comprises: (a) analyzing one or more polynucleotide sequences using an algorithm that determines whether the polynucleotide sequence is predicted to encode a polypeptide that is secreted from a cell in which the polypeptide is expressed; and (b) determining whether an mRNA that corresponds to one or more of the polynucleotide sequences that are predicted to encode secreted polypeptides is differentially expressed in one or more types of cancer cells compared to non-cancer cells.
- An mRNA that is differentially expressed in cancer cells compared to non-cancer cells, or a polypeptide encoded by the differentially expressed mRNA is useful as
- a biological sample e.g., serum
- a subject e.g., a mammal
- detecting in the biological sample an abnormal level of at least one secreted biomarker for that cancer.
- the secreted biomarkers for diagnosing prostate cancer can include relaxin 1 (HI), neuropeptide Y, MIC- 1, pancreatic thread protein-like (rat), prostate-specific membrane antigen, prostate-specific membrane antigen, prostate-specific membrane antigen, and single-minded homolog 2 (Drosophila).
- Figure 1 shows a schematic of a strategy for identifying genes that encode secreted proteins that are differentially expressed in cancer tissues.
- Figures 2A-2B show that expression of candidate secreted biomarkers are elevated in multiple cancer-types. Thirty-two genes encoding secreted proteins selected by annotation- and sequence-based analyses had significant overexpression in at least one tumor-normal counterpart tissue pair (>3-fold), and significant overexpression in tumors compared to any other normal tissue (>2-fold).
- BR- breast (ER+, ER-); CO - colorectal, GA - gastric/esophagus adenocarcinoma; KI - kidney, LI- liver; LUA - adenocarcinoma of the lung; LUS - squamous carcinoma of the lung; LUO - lung "other" - small cell lung carcinomas, large cell undifferentiated carcinomas of the lung; ON - ovary; PA - pancreas; PR -prostate.
- Figure 2A An expanded view of genes preferentially upregulated in carcinomas of the prostate is shown in Figure 2B.
- Figures 3 A-3B show validation of microarray gene expression by RT- PCR, IHC and ELISA.
- Figure 3 A shows RNAs expressed from multiple different human tissues (labeled above the RT-PCR panel), three normal, and six primary prostate carcinomas were reverse transcribed and amplified under standard conditions using primers directed toward relaxin-1. The primary microarray data is shown at top (hybridization intensity on the Y-axis; samples on the X-axis), and a representative PCR is shown at bottom. Primers specific for 18S were used to control for the amount of amplified cDNA.
- Figure 3B shows IHC performed with an anti-NPY antibody on whole tissue sections. Primary microarray data is shown at top, with examples of IHC staining in normal, microarray-positive and microarray-negative prostate cancers.
- Figures 4A-4C show validation of increases in the levels of candidate diagnostic proteins.
- Antibodies specific to candidate secreted proteins NPY (Fig. 4A), MUC-2 (Fig. 4B) and Maspin (Fig. 4C), were used to stain tissue microarrays containing 36 normal epithelial tissues and 229 carcinomas. The relative levels of expression for each gene are depicted at the top of each figure, with groups of tissues, and specific tissues identified. Gene expression levels were output in Tree View. Examples of IHC staining are shown at the bottom of each figure, highlighting both negative and positive cancers for each protein.
- Figures 5 A-5B show that upregulation of maspin expression correlates with estrogen receptor status in breast carcinomas.
- Fig. 5 A shows expression of maspin monitored by two independent probe-sets of the Affymetrix U95a GeneChip (PS1 and PS2).
- Fig. 5B shows comparison of microarray data and IHC on tissue microarrays in normal, ER+ and ER- tumors using an anti-maspin antibody. Note the intense staining on ER- tumors compared with normal ductal breast tissue
- Figure 6 shows expression of candidate lung cancer markers in an expanded set of normal and tumor lung tissue samples.
- the expression levels of lung cancer candidate genes (derived from each group of candidates - GO annotation and sequence- positive; GO annotation-positive only and sequence-positive only; Table 1) was determined in normal lung, lung adenocarcinomas, small cell undifferentiated carcinomas, squamous carcinomas and carcinoids. Data from the study (available at http://research.dfci.harvard.edu/meyersonlab/lungca/data.html) were downloaded and output in TreeNiew. Note the high levels of expression of GRP in carcinoids and SCLC, and the near-uniform overexpression of maspin in squamous carcinomas.
- the invention provides a global approach to the discovery of secreted, cancer-specific biomarkers.
- the methods involve identifying nucleic acids that encode proteins that are likely to be secreted from cells in which the proteins are expressed, and from that set of secreted protein-encoding nucleic acids, identifying those that exhibit differential expression in cancer cells compared to non-cancer cells.
- biomarkers that can be detected in samples obtained from a human or other mammal, thus facilitating the diagnosis of cancer in the mammal.
- the biomarker polypeptides are detectable in the blood, serum, or other biological sample that is readily obtainable from the mammal.
- Nucleic acids that encode secreted proteins can be identified using methods known to those of skill in the art.
- secreted proteins can be identified by virtue of an annotation associated with a nucleotide or amino acid sequence that is present, for example, in a database.
- suitable sources of such annotated sequences are the databases provided by the Genome Ontology Consortium (http://www.genomeontology.org). One can search the database for sequences that are annotated as encoding proteins that are found in cellular locations that are indicative of secretion.
- a suitable algorithm for this analysis is the "Tmap" program (Persson et al., JMol Biol, 237:182-192, 1994). This program is available on the internet at, for example, http://www.mbb.ki.se/tmap/.
- Secreted polypeptides can also be identified using an algorithm that identifies amino acid sequences within the polypeptide that comprise signal peptides, and/or that recognize cleavage sites for signal polypeptides.
- An example of software that conducts this type of analysis is "Sigcleave” (von Heijne et al., Nucl. Acids Res., 14:4683-4690, 1986).
- Sigcleave estimates the likelihood of an authentic signal peptide cleavage site in arbitrary amino acid sequence data. Sigcleave is available on the internet at, for example, http://bioweb.pasteur.fr/seqanal/interfaces/sigcleave.html.
- the set of polynucleotides that encode proteins that are predicted to be secreted from a cell are then subjected to expression analysis in tumor cells and non-tumor cells to identify those that exhibit differential expression in tumor cells compared to non- tumor cells.
- These secreted, differentially expressed polypeptides are suitable for use as biomarkers for the cancers in which the polypeptides are overexpressed.
- the level of expression of at least one of the genes that encode secreted polypeptides in the samples obtained from the subject and disease-free subject can be detected by measuring either the level of mRNA corresponding to the gene, the protein encoded by the gene or a fragment of the protein.
- the level of expression of one of the disclosed genes in a cancer tissue preferably differs from the level of expression of the gene in a non-cancer tissue by a statistically significant amount. In presently preferred embodiments, at least about a 2-fold difference in expression levels is observed. In some embodiments, the expression levels of a gene differ by at least about 3-, 5-, 10- or 100-fold or more in the cancer tissue compared to the non-cancer tissue.
- RNA can be isolated from the samples by methods well known to those skilled in the art as described, e.g., in Ausubel et al., Current Protocols in Molecular Biology, Vol. 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc. (1996).
- Methods for detecting the level of expression of mRNA are well known in the art and include, but are not limited to, northern blotting, reverse transcription PCR, real time quantitative PCR and other hybridization methods.
- a particularly useful method for detecting the level of mRNA transcripts obtained from a plurality of the disclosed genes involves hybridization of labeled mRNA to an ordered array of oligonucleotides.
- Such a method allows the level of transcription of a plurality of these genes to be determined simultaneously to generate gene expression profiles or patterns.
- the gene expression profile derived from the sample obtained from the subject can be compared with the gene expression profile derived from the sample obtained from the cancer-free subject to determine whether the genes are over- expressed in the sample from the subject relative to the genes in the sample obtained from the disease-free subject, and thereby determine whether the subject has or is at risk of developing a cancerous disorder (e.g., prostate cancer or colon cancer).
- a cancerous disorder e.g., prostate cancer or colon cancer
- the oligonucleotides utilized in this hybridization method typically are bound to a solid support.
- solid supports include, but are not limited to, membranes, filters, slides, paper, nylon, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, polymers, polyvinyl chloride dishes, etc.
- Any solid surface to which the oligonucleotides can be bound, either directly or indirectly, either covalently or non- covalently, can be used.
- a particularly preferred solid substrate is a high density array or DNA chip (e.g., the U95a GeneChipTM from Affymetrix Inc., Santa Clara, CA).
- These high density arrays contain a particular oligonucleotide probe in a pre-selected location on the array. Each pre-selected location can contain more than one molecule of the particular probe. Because the oligonucleotides are at specified locations on the substrate, the hybridization patterns and intensities (which together result in a unique expression profile or pattern) can be interpreted in terms of expression levels of particular genes.
- the oligonucleotide probes are preferably of sufficient length to specifically hybridize only to complementary transcripts of the above identified gene(s) of interest.
- the term "oligonucleotide” refers to a single-stranded nucleic acid. Generally the oligonucleotides probes will be at least 16 to 20 nucleotides in length, although in some cases longer probes of at least 20 to 25 nucleotides will be desirable.
- the oligonucleotide probes can be labeled with one or more labeling moieties to permit detection of the hybridized probe/target polynucleotide complexes.
- Labeling moieties can include compositions that can be detected by spectroscopic, biochemical, photochemical, bioelectronic, immunochemical, electrical optical or chemical means.
- labeling moieties include, but are not limited to, radioisotopes, e.g., 32 P, 33 P, 35 S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, linked enzymes, mass spectrometry tags, and magnetic labels.
- Oligonucleotide probe arrays for expression monitoring can be prepared and used according to techniques which are well known to those skilled in the art as described, e.g., in Lockhart et al., Nature Biotechnology, Vol. 14, pp. 1675-1680 (1996); McGall et al, Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 13555-13460 (1996); and U.S. Patent No. 6,040,138.
- tissue microarrays can be constructed according to methods routinely practiced in the art. For example, microarrays containing multiple tissue samples can be prepared using a Tissue Microarrayer (Beecher Instruments, Silver Spring, MD) with, e.g., zinc formalin-fixed, paraffin-embedded specimens. Each microarray can contain one core of each neoplasm whose transcripts are profiled in the analysis.
- a tissue microarray can comprise a set of tissues from different carcinomas, as well as cores of selected normal tissues (see the Examples below).
- antibody includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional antibody fragments, which are those fragments sufficient for binding of the antibody fragment to the protein or a fragment of the protein.
- Antibodies used in IHC (immunohistochemistry) analysis of the TMAs can be generated using methods well known and routinely practiced in the art. Some antibodies employed to practice the present invention can also be obtained commercially, e.g., monoclonal anti-MUC-2 (BioGenex, San Ramon, CA); monoclonal anti-maspin (Novocastra Laboratories, Newcastle upon Tyne, UK); and rabbit polyclonal anti- neuropeptide Y (Research Diagnostics, Inc, Flanders NJ).
- various host animals may be immunized by injection with the polypeptide, or a portion thereof.
- host animals may include, but are not limited to, rabbits, mice and rats, to name but a few.
- adjuvants may be used to increase the immunological response, depending on the host species, including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueri ) and Corynebacterium parvum.
- Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
- an antigen such as target gene product, or an antigenic functional derivative thereof.
- host animals such as those described above, may be immunized by injection with the encoded protein, or a portion thereof, supplemented with adjuvants as also described above.
- Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein (Nature, Vol. 256, pp. 495- 497 (1975); and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., Immunology Today, Vol. 4, p. 72 (1983); Cole et al., Proc. Natl. Acad. Sci. USA, Vol. 80, pp.
- Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.
- the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. [00034]
- techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
- a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable or hypervariable region derived from a murine mAb and a human immunoglobulin constant region.
- Antibody fragments which recognize specific epitopes may be generated by known techniques.
- such fragments include, but are not limited to, the F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule, and the Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
- Fab expression libraries may be constructed (Huse et al., Science, Vol. 246, pp. 1275-1281 (1989)) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
- immunoassay methods which utilize the antibodies described above.
- immunoassay methods include, but are not limited to, dot blotting, western blotting, competitive and noncompetitive protein binding assays, enzyme-linked immunosorbant assays (ELISA), immunohistochemistry, fluorescence-activated cell sorting (FACS), and others commonly used and widely described in scientific and patent literature, and many employed commercially.
- sandwich ELISA of which a number of variations exist, all of which are intended to be encompassed by the present invention.
- unlabeled antibody is immobilized on a solid substrate and the sample to be tested is brought into contact with the bound molecule and incubated for a period of time sufficient to allow formation of an antibody-antigen binary complex.
- a second antibody labeled with a reporter molecule capable of inducing a detectable signal, is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labeled antibody.
- any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.
- Variations on the forward assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse assay, in which the labeled antibody and sample to be tested are first combined, incubated and added to the unlabeled surface bound antibody.
- reporter molecules in this type of assay are either enzymes, fluorophore- or radionuclide-containing molecules.
- an enzyme is conjugated to the second antibody, usually by means of glutaraldehyde or periodate.
- glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different ligation techniques exist which are well-known to the skilled artisan.
- Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, among others.
- the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change.
- p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product, rather than the chromogenic substrates noted above. A solution containing the appropriate substrate is then added to the tertiary complex. The substrate reacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an evaluation of the amount of secreted protein or fragment thereof, e.g., PLAB or the catalytic domain of hepsin, which is present in the serum sample.
- fluorogenic substrates which yield a fluorescent product
- fluorescent compounds such as fluorescein and rhodamine
- fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
- the fluorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic longer wavelength. The emission appears as a characteristic color visually detectable with a light microscope.
- Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.
- the present invention provides methods for diagnosing various forms of cancers or a predisposition to develop any of the cancers.
- the methods comprise detecting at least one (e.g., 1, 2, 3, 4, 5, or more) differentially expressed cancer-specific biomarkers for a given cancer that have been identified in accordance with the present invention (e.g., see Table 1).
- a diagnostic test works by comparing a measured level of at least one biomarker (e.g., MIC-1) in a subject (e.g., a mammal) with a baseline level determined in a control population of subjects unaffected by cancer.
- abnormal expression of a biomarker is limited to a specific tissue type (e.g., breast tissue for breast cancer).
- the baseline level of the biomarker for comparison can also be an expression level of the biomarker in control tissues where the cancer is not present.
- the measured level does not differ significantly from baseline levels in a control population (or control tissues), the outcome of the diagnostic test is considered negative.
- the outcome of the diagnostic test is considered negative.
- the subject if there is a significant departure between the measured level in a subject and baseline levels in unaffected subjects (or control tissues), it signals a positive outcome of the diagnostic test, and the subject is considered to have abnormal presence or an abnormal level of that biomarker.
- the departure is an increase in expression levels of the biomarkers.
- abnormality can also be a decreased expression level.
- a departure from baseline levels is statistically significant if at least a 2-fold difference in expression levels is observed.
- a departure with less than a 2-fold difference in the expression levels can still be considered significant, if the measured value falls outside the range typically observed in unaffected subjects due to inherent variation between subjects and experimental error.
- a departure can be considered significant if a measured level does not fall within the mean plus one standard deviation of levels in a control population.
- a significant departure may occur if the difference between the measured level and baseline levels is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
- the extent of departure between a measured value and a baseline value in a control population also provides an indicator of the probable accuracy of the diagnosis, and/or of the severity of the disease being suffered by the subject.
- the methods for diagnosing cancers can entail obtaining from a subject an expression profile of biomarkers for a given cancer, and comparing the gene expression profile to at least one expression profile from subjects known to have the cancer.
- the profile can contain expression levels (e.g., in the serum) of at least one (e.g., 1, 2, 3, 4, 5, or more) biomarkers for that cancer.
- Methods of obtaining expression profiles and their uses in disease diagnosis are well known in the art.
- methods of the present invention can be practiced using the specific biomarkers identified by the present inventors with techniques described in, e.g., USPN 6,365,352 or WO0111082.
- a preferred biological sample for measuring levels of the secreted cancer biomarkers is serum.
- Other tissue samples from blood e.g., whole blood and plasma, may also be used to measure levels of the secreted biomarkers in a subject and the- control population.
- Other than blood related biological samples other samples may also be employed for measuring expression levels of the cancer biomarkers. These include, e.g., samples obtained from any organ, tissue, or cells, as well as urine, or other bodily fluids.
- the sample can be an tissue biopsy obtained from skin, hair, urine, saliva, semen, feces, sweat, milk, amniotic fluid, liver, heart, muscle, kidney and other body organs. Tissue samples are typically lysed to release the protein and/or nucleic acid content of cells within the samples. The protein or nucleic acid fraction from such crude lysates can then be subject to partial or complete purification before analysis.
- Examples of these secreted biomarkers that are suitable for diagnosing cancers are set forth in Table 1 below.
- some methods for diagnosing the existence of, or a predisposition to develop, prostate cancer can comprise detecting differentially expressed levels of relaxin-1, MIC-1, or neuropeptide Y.
- detection of differentially expressed MUC-2 may lead to diagnosis of colon cancer.
- some methods of the invention are directed to diagnosing cancers in several tissues. For example, detection of a differentially expressed level of mapsin or MUC-1 can indicate the existence of, or a predisposition to develop, a cancer in the prostate, colon, or other tissues (see Examples below).
- the methods can further comprise examining a subject with a conventional procedure for detecting and diagnosing cancers.
- a conventional procedure for detecting and diagnosing cancers Such procedures are well known and routinely practiced in the art, e.g., CAT scanning, MRI, and ultrasonography.
- Other procedures for diagnosing various forms of cancers are also described in the art, e.g., at ht1p://www.bccancer.bc.ca/PPI/TypesofCancer/CancerinGeneral/DiagnosingCancer.
- Methods of the present invention are suitable for large scale screening of a population of subjects for the presence or a predisposition to the development of the various forms of cancers.
- the methods can be employed in conjunction with additional biochemical and/or genetic markers of other disorders that may reside in the subjects.
- kits for detecting the level of expression of at least one biomarker identified using the methods of the invention.
- the kit can comprise a labeled compound or agent capable of detecting a protein encoded by, or mRNA corresponding to, at least one of the biomarkers, means for determining the amount of protein encoded by or mRNA corresponding to the gene or fragment of the protein; and means for comparing the amount of protein encoded by or mRNA corresponding to the gene or fragment of the protein, obtained from the subject sample with a standard level of expression of the gene, e.g., from a cancer-free subject.
- the compound or agent can be packaged in a suitable container.
- the kit can further comprise instructions for using the kit to detect protein encoded by or mRNA corresponding to the gene.
- the invention also provides methods that are suitable for monitoring subjects who have previously been diagnosed with a cancer, particularly their response to treatment.
- progression of a cancer in a subject can be monitored by measuring a level of expression of a biomarker identified using the methods of the invention, in a sample of bodily fluid or other tissue obtained in the subject over time, i.e., at various stages of the cancer.
- An increase in the level of expression of the mRNA or encoded protein corresponding to the gene(s) over time is indicative of the progression of the disorder (e.g., prostate cancer or colon cancer).
- the level of expression of mRNA and protein corresponding to the gene(s) can be detected by standard methods as described above.
- Example 1 Identification of genes encoding secreted proteins
- This Example describes an example of the use of the invention to identify biomarkers for cancer.
- the expression of ⁇ 12,500 transcripts was surveyed in a series of 45 normal and 150 malignant tissue samples representing carcinomas of the prostate, breast, lung, ovary, colorectum, kidney, liver, pancreas, bladder/ureter and stomach /esophagus.
- a combination of database annotations and predicted amino acid sequence analysis identified a subset of 576 genes that predominantly encode secreted proteins, of which 32 exhibited cancer-specific overexpression.
- Several of the identified genes encode known or candidate diagnostic proteins, such as mammaglobin in breast cancer, and kallikreins 6 and 10 in ovarian cancer, respectively.
- Oligonucleotide probe-sets were filtered for candidate genes encoding secreted proteins by two distinct approaches, as shown in Figure 1.
- Figure 1A probe-sets were mapped to Genome Ontology (GO) Consortium annotations (www.genomeontology.org), and those with "location” annotations suggesting protein secretion were identified (1,160).
- Figure IB protein sequences of the genes represented on the oligonucleotide microarray were interrogated using two sequence-based algorithms, “tmap” (Persson et al., JMol Biol, 237:182-192, 1994) and "sigcleave” (von Heijne et al., Nucl.
- the 30 terms are blood coagulation; blood coagulation factor; cell-cell signaling; cell communication; complement activation; complement component; diuretic hormone; ephrin; extracellular; extracellular matrix; extracellular matrix glycoprotein; extracellular matrix structural protein; extracellular space; hormone; insulinlike growth factor receptor ligand; interleukin 12 receptor ligand; interleukin 2 receptor ligand; interleukin 4 receptor ligand; interleukin 5 receptor ligand; interleukin 6 receptor ligand; interleukin 7 receptor ligand; interleukin 8 receptor ligand; leukemia inhibitor factor receptor ligand; ligand; neuropeptide hormone; opsonin; protein secretion; secreted phospholipase A2; tissue kallikrein; vascular endothelial growth factor receptor ligand.
- Example 2 Expression of candidate genes in tumors [00060] This example describes overexpression of candidate genes encoding secreted proteins in tumors of diverse anatomic origin. Expression of these 2,308 genes was examined in a series of 150 carcinomas representing 10 distinct anatomic origins, 46 normal tissues from the corresponding anatomic sites, and nine other anatomic sites not represented in our "tumor/normal" collection (Fig. 2). What were so ⁇ ght are genes whose expression was high in tumors of one or more sites of origin, with correspondingly low or absent expression in other normal body tissues.
- TMAs tissue microarrays
- Example 3 Validation of candidate genes in tumor samples [00065] Validation of the above approach first comes from the observation that many of the genes identified here encode secreted proteins previously shown to be dysregulated in cancer tissue (e.g., by other transcript-based approaches, or by IHC), or shown to be elevated in the serum from cancer patients compared to matched controls.
- GRP/bombesin gastrin-releasing peptide
- lung carcinomas Heasley, Oncogene 20, 1563-9, 2001
- KLK6, KLK10 kallikreins 6 and 10
- AFP alpha-fetoprotein
- MGBA mammaglobin A
- Table 1 Genes encoding candidate secreted proteins overexpressed in carcinomas
- TNF alpha-induced protein 6 NM_007115 GO and TMSC Kidney 103 4 5
- Indian hedgehog homolog (Drosophila) none GO only Gastric 4.7 2.4 adipose differentiation-related protein NM_001122 GO only Kidney 9.6 2.6 vascular endothelial growth factor receptor NM_002019 GO only Kidney 3.8 2.4 alpha-fetoprotein NM_001134 GO only Liver 7.6 7.6 hypocretin (orexin) receptor 1 NM_001525 GO only Liver 5.4 5.4 interleukin 1, beta NM_000576 GO only Lung, other 33.5 33.5 neurotensin receptor 1 (high affinity) NM_002531 GO only Lung, other 10.3 10.3 plasminogen activator inhibitor type 1 NM_000602 GO only Lung, other 43.4 5.4 ephrin-B2 NM_004093 GO only Lung, other 3.5 2.8 interleukin 1, beta NM_000576 GO only Lung, other 3.1 2.2 epiregulin NM_001432 GO only L
- TNF-r superfamily member 17 NM_001192 TMSC Only Liver 4.2 3.1 bone morphogenetic protein 6 NM_001718 TMSC Only Lung, AdCa 3.1 3.1
- TSS candidate 3 NM_003311 TMSC Only Lung, other 7.1 2.4 transcription factor A, mitochondrial M_003201 TMSC Only Lung, other 3.4 2.1 lymphocyte antigen 6 complex, locus D NM_003695 TMSC Only Lung, SCC 50.7 14.5 ovalbumin NM_002639 TMSC Only Lung, SCC 68.1 5.5 ovalbumin NM_002639 TMSC Only Lung, SCC 29.3 3.4
- T/CN ratio of expression of tumor tissue and corresponding normal tissue
- T/OtherN ratio of expression of tumor tissue and "other" normal tissues
- Lung, other lung carcinomas with histologies other than squamous or adeocarcinoma
- NPY neuropeptide Y
- tissue microarrays containing 229 carcinomas and 36 normal tissues samples of diverse anatomic origin were stained with a commercial anti-NPY antibody.
- staining was found in nerves and a few prostate secretory epithelial cells, while in prostate carcinomas that had high NPY gene expression, correspondingly high levels of protein expression were found (Fig. 3B).
- the anti-NPY antibody did not stain carcinomas of other anatomic sites on the TMAs, nor other non-neural or non-neuroendocine normal tissues that were included on these arrays.
- the instant example describes candidate gene expression in other independent datasets.
- publicly accessible data from Bhattacharjee et al. enabled analysis of expression of the lung cancer candidate genes of the present invention in 203 lung tissues, including 17 samples of normal lung, 127 adenocarcinomas, 21 squamous carcinomas, 20 pulmonary carcinoids, and 6 small cell undifferentiated carcinomas. This analysis demonstrated some striking correlations of gene expression with histological subtype.
Abstract
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US8321137B2 (en) * | 2003-09-29 | 2012-11-27 | Pathwork Diagnostics, Inc. | Knowledge-based storage of diagnostic models |
US20050069863A1 (en) * | 2003-09-29 | 2005-03-31 | Jorge Moraleda | Systems and methods for analyzing gene expression data for clinical diagnostics |
WO2005103069A1 (en) * | 2004-04-19 | 2005-11-03 | University Of Florida Research Foundation, Inc. | Multidimensional protein separation |
ES2543985T3 (en) * | 2007-10-22 | 2015-08-26 | St Vincent's Hospital Sydney Limited | Forecast Methods |
ES2434996T3 (en) | 2008-10-31 | 2013-12-18 | St Vincent's Hospital Sydney Limited | Prognosis methods in chronic kidney disease |
EP2910650A1 (en) | 2009-08-24 | 2015-08-26 | National University Corporation Kanazawa University | Detection of colorectal cancer by gene expression profiling |
WO2011050407A1 (en) * | 2009-10-28 | 2011-05-05 | St Vincent's Hospital Sydney Limited | Methods of diagnosing and prognosing colonic polyps |
WO2011096698A2 (en) * | 2010-02-05 | 2011-08-11 | 국립암센터 | Composition for cancer prognosis prediction comprising anti-tmap/ckap2 antibodies |
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